Molecular Crosstalk between RNA-binding Proteins and microRNA in the Translation Regulation of p53 mRNA in Response to DNA Damage

Goswami, Binita (2023) Molecular Crosstalk between RNA-binding Proteins and microRNA in the Translation Regulation of p53 mRNA in Response to DNA Damage. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Binita Goswami (17RS065)) 17RS065.pdf - Submitted Version Restricted to Repository staff only Download (6MB)

Abstract

Post-transcriptional regulation of p53, by the microRNA miR-125b and the RNA-binding protein HuR, controls p53 expression under genotoxic stress. p53 mRNA translation is repressed by miR-125b, tightly regulating its basal level of expression, whereas HuR prevents miR-125b binding to p53 mRNA and enhances p53 protein synthesis. The translation repression of p53 mRNA is relieved upon DNA damage by a decrease in miR-125b level, contributing to the pulsatile expression of p53. The pulse of p53, as also of HuR, in response to UV irradiation coincides with a time-dependent biphasic change in miR-125b level, opposite in phase to the p53 and HuR pulses. This thesis investigates the molecular mechanisms underlying the biphasic change in the miR-125b level and their effect on the cellular regulatory network controlling p53 expression in response to UV-induced DNA damage. We show that the cause for the decrease in miR-125b level immediately post DNA damage is enhanced exosomal export mediated by HuR. The subsequent increase in miR-125b level is due to p53-mediated transcriptional upregulation and enhanced processing, demonstrating miR-125b as a transcriptional and processing target of p53. p53 activates the transcription of primary miR-125b RNA from a cryptic promoter in response to UV irradiation. The resultant enhanced level of miR-125b represses p53 mRNA translation, bringing p53 protein down to the basal level. Together, these regulatory processes constitute reciprocal feedback loops that determine the biphasic change in miR-125b level, ultimately contributing to the fine-tuned temporal regulation of p53 expression in response to genotoxic stress. We have further expanded the understanding of the regulatory network of p53 by investigating the role of Wig-1, a p53-induced RBP which regulates the stability and translation of p53 mRNA in response to UV irradiation. Using nuclear magnetic resonance (NMR) and microtubule bench (MT-bench)-based approaches, we have demonstrated the interaction of Wig-1 with RNA and with Ago2, the central component of the RNA-induced silencing complex (RISC). Wig-1 appears to prevent miR-125b-mediated translation repression of p53 mRNA under DNA damage, possibly by interacting with Ago2 and displacing the miRISC complex from the p53 mRNA. Therefore, the concerted action of the two RBPs, HuR and Wig-1, and the interplay with the miRNA miR-125b, works via multiple molecular mechanisms in the regulatory network controlling p53 expression in response to DNA damage.

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